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A general route to nanostructured M[V3O8] and M-x[V6O16] (x=1 and 2) and their first evaluation for building enzymatic biosensors

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A general route to nanostructured M[V3O8] and

M-x[V6O16] (x=1 and 2) and their first evaluation for

building enzymatic biosensors

Nathalie Steunou, Christine Mousty, Olivier Durupthy, Cécile Roux,

Guillaume Laurent, Corine Simonnet-Jégat, Jacky Vigneron, Arnaud

Etcheberry, Christian Bonhomme, Jacques Livage, et al.

To cite this version:

Nathalie Steunou, Christine Mousty, Olivier Durupthy, Cécile Roux, Guillaume Laurent, et al.. A

general route to nanostructured M[V3O8] and M-x[V6O16] (x=1 and 2) and their first evaluation for

building enzymatic biosensors. Journal of Materials Chemistry, Royal Society of Chemistry, 2012, 22

(30), pp.15291-15302. �10.1039/c2jm30485f�. �hal-01461422�

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A general route to nanostructured M[V

3

O

8

] and M

x

[V

6

O

16

] (

x ¼ 1 and 2) and

their first evaluation for building enzymatic biosensors†

Nathalie Steunou,*

ab

Christine Mousty,

c

Olivier Durupthy,

a

Cecile Roux,

a

Guillaume Laurent,

a

Corine Simonnet-Jegat,

b

Jacky Vigneron,

b

Arnaud Etcheberry,

b

Christian Bonhomme,

a

Jacques Livage

a

and Thibaud Coradin

a

Received 25th January 2012, Accepted 16th May 2012 DOI: 10.1039/c2jm30485f

In order to develop novel electroactive hosts for biosensor design, the possibility to use nanostructured vanadate phases as alternatives to well-known V2O5gels was studied. For this purpose, the formation of M[V3O8] and Mx[V6O16] (x¼ 1 and 2) oxides by the sol–gel process has been studied over a wide range of cations (M+¼ Li+, Na+, K+, Cs+, and NH

4+; M2+¼ Ca2+, Mg2+and Ba2+). By a combination of XRD,51V NMR and SEM studies, it was possible to evidence the influence of the nature and hydration

state of cations on the size and morphology of the resulting particles as well as on the kinetics of their formation. On this basis, K2[V6O16] was evaluated for glucose oxidase encapsulation, either via impregnation or co-precipitation methods. When compared to V2O5, these novel bioelectrodes exhibit higher stability under pH conditions of optimum enzymatic activity, as well as better sensitivity, and reproducibility for glucose detection via amperometric titration.

1.

Introduction

There is a growing interest towards functional bionanocompo-sites that act as the active part of an electrochemical, optical or photoelectrical device.1–4 In particular, cells and enzymes with specific catalytic properties have been combined to inorganic and bioorganic matrices for the building-up of biosensors, bioreac-tors or biofuel cells.5 Since the performance of a

bio-electrochemical device primarily depends on the stability (under use and storage) of the immobilized biomacromolecules within the matrix and the detection of a measurable signal, current developments focus on new immobilization materials.6 Until recently, bioencapsulation in inorganic hosts has mainly been

performed in silica,6–8 layered double hydroxides and clay minerals9 due to their natural origin and/or well-established

biocompatibility. In most cases, the function of these inorganic matrices is to preserve the tertiary structure of the bio-macromolecules and to avoid their denaturation.7 However, it has been recently shown that interfacing biomacromolecules with inorganic solids can be successfully extended to non-biogenic phases such as metallic particles and metal oxides.10–13Actually,

these phases exhibit interesting physical properties that may improve electrochemical systems or allow the design of new bionanotechnological devices.1,2,14,15 In particular, inorganic phases that exhibit semi-conducting properties have been used to build-up DNA-biochips16 or neuron-chips.17 In the same way,

one of the critical challenges in the building-up of biosensors and biofuel cells is to promote a direct electron transfer between enzyme and electrodes, which is difficult to achieve because of the location of the prosthetic group within the surrounding protein matrix and consequently the kinetic barrier for electron transfer resulting from the distance between the redox active site and the electrode surface. Since most of the host matrices used so far are non-conductive, the co-insertion of redox mediators or metallic particles within the immobilization matrix is often required.6,9a These species can either catalyze a redox reaction with a substrate or shuttle the electrons more efficiently between enzyme and electrode. An alternative promising approach would consist in the encapsulation of enzymes in an electroactive matrix, providing the means for building mediator-free biosen-sors or third generation biosenbiosen-sors.9a,18For most studied bio-electrodes, the electroactive inorganic phase may act as an electron relay to the electrode and thus enhances the electrical aChimie de la Matiere Condensee de Paris, UMR CNRS 7574, UPMC

Paris 6, College de France, 11 place Marcelin Berthelot, 75231 Paris Cedex 05, France

bInstitut Lavoisier, UMR CNRS 8180, UVSQ, 45 avenue des Etats-Unis,

78035 Versailles Cedex, France. E-mail: nathalie.steunou@uvsq.fr; Fax: +33 1 39 25 43 73; Tel: +33 1 39 25 44 52

cInstitut de Chimie de Clermont-Ferrand ICCF, CNRS UMR 6296,

Universite Blaise Pascal, 63177 Aubiere Cedex, France

† Electronic supplementary information (ESI) available: XRD patterns (Fig. S1) of (NH4)2[V6O16]$H2O, (NH4)2[V6O16]$1.5H2O, Ca[V6O16]$

9.7H2O, Mg[V6O16]$7.1H2O, and Ba1.2[V6O16]$5.5H2O; SEM images of

M[V6O16]$nH2O (with M2+ ¼ Mg2+, Ca2+, and Ba2+) (Fig. S2) and

K2[V6O16]–GOx biomembrane (Fig. S3); amount of tri- and

hexavanadate phases versus pH (Fig. S4); the XRD pattern of V2O5$nH2O at pH values of 1, 4 and 5 (Fig. S5); FT-IR spectra of

GOx and K2[V6O16] (Fig. S6); XPS spectra of K2[V6O16] – pH 3,

K2[V6O16] – pH 6, K2[V6O16]–GOx (Fig. S7 and S8), and V2p peak

positions (Table S1); storage stability of K2[V6O16]–GOxcos (Fig. S9).

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signal of the transducer leading to an improved sensitivity and a lower detection limit of analytes. Currently, the most studied methodology relies on the preparation of composite materials where a conductive network of particles over the percolation threshold (for instance carbon nanotubes) is trapped within a non-conductive matrix, mainly a bio-organic polymer19 or a sol–gel matrix.20 Alternatively, it should be possible to use single-phase, conductive oxides obtained via the sol–gel process as hosts for enzymes. However, the adaptation of encapsulation procedures developed for silica over the last few years is not straightforward, due to different reactivities (pH, solvent, redox,.) both at the stage of preparation and also in terms of working conditions. This probably explains why non-silica sol–gel based matrices were scarcely employed to entrap enzymes.18a,b,e,21Among the potential host candidates, V

2O5gels appear to be very interesting due to their conductive, catalytic and intercalation properties that have been widely studied over the last 30 years.22 However, V2O5 is easily reduced by many organic molecules and stable only under acidic conditions, explaining why only two previous reports were devoted to V2O5– glucose oxidase (GOx) biosensors.23,24 In the field of

electro-chemical devices, the initial interest in V2O5materials has rapidly shifted to other V(V) phases, such as M[V3O8] and Mx[V6O16] (x¼ 1 and 2) (see Fig. 1) for the building-up of positive electrodes for Li-batteries.25They were mainly prepared in the literature

through hydrothermal and solid state routes. However, numerous studies have demonstrated the strong influence of the grain morphology and the texture of the material on the elec-trochemical performances of Li1+a[V3O8].25Since the synthesis of materials through low temperature chemical routes allows a better control of their microstructure and texture, we developed recently a simple sol–gel method for synthesizing nanostructured M[V3O8] with monovalent cations M+¼ Na+26and Cs+.27

In the present work, we hypothesized that these phases may also be advantageously developed as an alternative to V2O5for enzyme encapsulation and biosensor design. In a first step, we have tried to extend our previous method to a wide range of cations including divalent ones, to identify the most suitable host for further enzyme encapsulation. An attempt was made to rationalize the effect of the cation size and hydration on the kinetics of formation and structure of the resulting materials. This control over the nucleation and growth of vanadium oxides appears to be crucial for designing new bioelectrochemical devices such as enzymatic biosensors since the control of porosity (organization, pore size, and connectivity) and texture in sol–gel coatings can modulate the enzyme immobilization, and enhance the diffusion of electroactive species, analytes and reaction products. In the second step, we have used (and for the first time to our knowledge) a nanostructured K2[V6O16]$nH2O material to encapsulate glucose oxidase (GOx) as a model enzyme and the resulting bionanocomposites were applied to the electrochemical detection of glucose. These GOx–K2[V6O16] biosensors showed superior stability and analytical performance compared to V2O5-based systems. The GOx–K2[V6O16] biosensors present interesting and very promising properties in terms of sensitivity, reproducibility and response time, showing that vanadium oxides may presumably compete with other inorganic matrices to entrap enzymes. These results raise hope for the future development of third-generation biosensors that would benefit

from the chemical, structural and functional versatility of V(V)-based materials.

2.

Experimental section

2.1 Synthesis of M[V3O8] and Mx[V6O16] (x ¼ 1 and 2) M[V3O8] and Mx[V6O16] (x ¼ 1 and 2) phases (with M+ ¼ Na+, K

+, Cs+ and NH

4+; M2+ ¼ Ca2+, Mg2+ and Ba2+) have been prepared via the acidification of an aqueous solution of sodium metavanadate NaVO3 (Sigma, >99%, 0.5 mol L1, pH ¼ 8)

according to the procedure used for the synthesis of vanadium oxide gels.28Acidification of a 0.5 mol L1metavanadate solution

(NaVO3), with an initial pH close to 8, was performed using a proton exchange resin (DOWEX 50WX 4–100 mesh). After complete proton exchange, a clear yellow solution (pH 1) is obtained that progressively turns into a red V2O5$nH2O gel after 12 h in the absence of additives. For the synthesis of M[V3O8] and M2[V6O16] phases, small volumes of MOH and MCl solu-tions (2 mol L1) were added to 10 mL of the yellow acidic solution of V(V) precursors in order to set the cation concen-tration to 0.2 mol L1and the initial pH (pH

i) to a precise initial value ranging from 1 to 4. The same procedure is used with M(OH)2and MCl2but the concentration of divalent cations is fixed to 0.1 mol L1. These solutions were then left for ageing at

40C for a period between one day and three weeks. Precipita-tion was observed for all investigated condiPrecipita-tions. Table 1 pres-ents the chemical composition of MxV2O5intercalates, M[V3O8] and Mx[V6O16] (x¼ 1 and 2) phases depending on the nature of cation and pHi, and according to chemical and thermogravi-metric analyses and powder X-ray diffraction.

2.2 Synthesis of GOx–K2[V6O16] biomembranes

Glucose oxidase (GOx) (E.C. 1.1.3.4. type VII from Aspergillus niger, 150–218 U mg1) was purchased from Sigma. The immo-bilization of GOx has been performed through a two step adsorption and a co-sedimentation method. For both methods, V2O5sols were first prepared by redispersion of V2O5$nH2O gels in distilled water of pH 5 and homogeneous suspensions of K2[V6O16]$nH2O (n¼ 1.5 and 2.7) were obtained by keeping the aqueous precipitate in an ultrasonic bath for 30 min. Coatings on glass or Pt electrode were prepared by solvent casting from the

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V2O5$nH2O sols and K2[V6O16]$nH2O suspensions and after drying the coatings for about 12 h, a small volume of GOx (10 mg mL1) in phosphate buffer was then deposited on these

films and dried. In order to enhance the lifetime of the bio-membrane due to a slow release of enzymes into solution, enzyme cross-linking with glutaraldehyde vapor (GA, 15 min) was per-formed. The alternative co-sedimentation method consists of mixing together the vanadium oxides and GOx solutions and spreading the aqueous mixture on a substrate. This co-sedi-mentation method avoids a possible leaching of entrapped enzymes from the biomembranes to the solution and the chem-ical reticulation step can be skipped. For both immobilization methods, the bioelectrodes are then washed in acetate (V2O5and K2[V6O16]) or phosphate (K2[V6O16]) buffers in order to remove the free enzymes before being used as biosensors. The prepara-tion of the Pt modified electrodes is summarized in Table 2, the deposited enzyme amount was fixed at 50mg.

2.3 Characterization

X-ray powder diffraction patterns were recorded on a Philips PW 1830 diffractometer using CuKa radiation (l ¼ 1.542 A). The FT-IR spectra were recorded on a Nicolet 6700 FT-IR using an Attenuated Total Reflection Infrared (ATR-IR) experiment at a resolution of 4 cm1. Scanning electron microscopy (SEM)

studies were performed on a Cambridge stereoscan 120 micro-scope using gold-coated samples. Thermogravimetric analyses (TGA) were performed on a TA SDT 2960 apparatus. Solids were heated up to 600C with a heating rate of 5C min1in an

oxygen atmosphere. The amount of V(IV) in the solids was measured by the spectroscopic UV-visible method according to

Table 1 Experimental conditions (pHiand ageing time), chemical composition of (a) MxV2O5$nH2O and (b) M[V3O8], Mx[V6O16] (x¼ 1 and 2) phases

Cation riona(A) pHib Chemical compositionc d001d(A) JCPDS/ref.

Li+ 0.74 1–2 Li 0.2V2O5$2.1H2O 12.8 45 Na+ 0.95 1–2 Na 0.3V2O5$1.5H2O 10.4 45 and 46 K+ 1.38 1 K 0.4V2O5$1.2H2O 10.3 45 and 46 Cs+ 1.70 3–4 4Cs[H 2V10O28]$4H2O — 80-0431 and 27,36 TMA+ 3.01 1–2 TMA 0.27V2O5$1.3H2O 12.3 45

3 4TMA[H2V10O28]$H2O — 51V MAS and 39

NH4+ 1.47 1–2 (NH4)0.3V2O5$1.5H2O 11.3 45 and 46 Mg2+ 0.72 1 Mg 0.15V2O5$2.4H2O 13.8 45 Ca2+ 0.99 1–2 Ca 0.15V2O5$2.0H2O 13.8 45 Ba2+ 1.36 1 Ba 0.15[V2O5]$1.5H2O 12.3 45 2–4 3Ba[V10O28]$21H2O 87-1902

Cation pHi Ageing time (days) Chemical composition JCPDS/ref.

Na+ 2 21 Na[V 3O8]$1.5H2O 16-0601 and 26 3–4 4 Na[V3O8]$1.5H2O 16-0601 and 26 K+ 2–4 1 K 2[V6O16]$2.7H2O 51-0379 K2[V6O16]$1.5H2O 54-062 Cs+ 1–2 1 Cs 2[V6O16] 84-2497 and 27 3 21 Cs2[V6O16]$0.7H2O 51-0378 and 27 3–4 4 Cs2[V6O16] 84-2497 and 27 NH4+ 2–4 4 (NH4)2[V6O16]$H2O 41-0492 3 1 (NH4)2[V6O16]$1.5H2O 51-0376 Mg2+ 2–3 21 Mg[V 6O16]$7.1H2O 46-0281 Ca2+ 2–4 4 Ca[V 6O16]$9.7H2O 35-0564 Ba2+ 2 14 Ba 1,2[V6O16]$5.5H2O 51-0375 a

Ionic radius.bInitial pH.cPhase obtained after 24 h.dBasal distance.

the previous study.26 X-ray photoelectron spectroscopy (XPS)

surface chemical analyses are performed with a Thermo Electron K-Alpha spectrometer with a base pressure of 5 1010Torr and using the AlKa (1486.5 eV) X-ray monochromatized radiation with a pass energy of 20 eV. Energy levels of XPS were calibrated with Au single crystal. The spectra were processed using the Thermo Advantage data system.51V MAS NMR spectra were

recorded at 79.0 MHz on a Bruker Avance 300 spectrometer using a 4 mm MAS Bruker probe. Solid samples were spun both at 10 and 14 kHz using ZrO2rotors.51V MAS NMR spectra were acquired with a rotor synchronized echo sequence (q–s–2q–s-acq. with q ¼ p/16, s ¼ 1/nr, where nris the spinning frequency) and with power levels corresponding to p/2 lengths for the liquid standard (NH4VO3) of approximately 2.5ms. A spectral width of 1 MHz and 0.5 s of recycle time were used. Short pulse widths were used to collect a maximum of spinning sidebands with undistorted intensity. An accumulation of 14 000 transients was usually performed on each sample. Isotropic chemical shifts were reported to VOCl3using a solution of 0.1 mol L1NH4VO3 (d ¼ 578 ppm) as the secondary reference. The effect of1H CW

decoupling on the resolution of the51V MAS NMR spectra was

checked. However, no difference in the spectral resolution was observed with or without1H decoupling even for the hydrated

vanadium oxides. As a consequence, 51V MAS NMR spectra

were acquired without1H decoupling. Numerical simulations of

the51V MAS NMR spectra were performed with the DMFIT

program including the QUASAR extension.29,30These

simula-tions included the effects of the chemical shift anisotropy, CSA, as well as first and second-order quadrupolar interactions. Both central and satellite transitions were considered. Several NMR parameters describing the interactions have been determined, including the quadrupole coupling constant (CQ), the quad-rupolar asymmetry parameter (hQ), the isotropic chemical shift (diso), the CSA (DdCSA) and the CSA asymmetry parameter (hCSA). Chemical shift tensor parameters diso,DdCSAand hCSA are defined as diso¼ (d11+ d22+ d33)/3,DdCSA¼ (d33 diso) and

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hCSA¼ðd33ðd22 disoÞ d11Þ. The quadrupolar parameters are defined as

CQ¼eQV33

h and hQ¼

V22 V11

V33 . The Euler angles, 4, c, and j, between CSA and quadrupolar tensors were estimated through the careful analysis of both isotropic lines and spinning side-bands. The51V MAS NMR spectra were fitted with a number of

40 spinning sidebands (SSBs) provided by DMFIT. 2.4 Amperometric titration of glucose

Stock solutions of glucose were allowed to mutarotate at room temperature. Enzymatic activity of immobilized GOx within bionanocomposites casted on glass slides was first verified by measuring the consumption of O2 in different buffer solutions containing 50 mmol L1 glucose using a Clark electrode.

Secondly, the amperometric measurements were carried out with a potentiostat EA161 (EDAQ) connected to a thermostatted cell with a three-electrode system. The working electrodes were platinum electrodes (diameter 5 mm) polished with 1 mm dia-mond paste and 0.05mm alumina before use. A Pt wire was used as the counter electrode and all potentials were measured relative to an Ag/AgCl saturated KCl electrode. All measurements were carried out at 30 C under stirring conditions using a rotating disk electrode from Radiometer (500 rpm). The glucose concentration was increased stepwise by adding in the 20 mL buffer (acetate or phosphate) solution defined volumes of concentrated analyte stock solution. Calibration curves were therefore obtained by measuring the oxidation current of hydrogen peroxide at Eapp¼ 0.7 V.

3.

Results and discussion

3.1 Sol–gel synthesis and characterisation of M[V3O8] and Mx[V6O16] (x ¼ 1 and 2) phases

The synthesis of trivanadate and hexavanadate phases is based on a room temperature process previously reported for the synthesis of sodium and cesium trivanadate (i.e. Na[V3O8]26and Cs[V3O8]27). In the present work, this procedure was generalized to a large range of monovalent (M+¼ Li+, Na+, K+, Cs+, NH

4+,

Table 2 Amperometric responses to glucose of different metal oxide based biomembranes

Matrix Imm.a(pH) Q ¼ G/Vb Sensitivityc(mAM1cm2) R2(n) K2[V6O16]$nH2O (n¼ 1.5 &2.7) Ads (5) 0.33 25 0.9994 (12) Ads (6) 0.16 48 0.9995 (12) Cos (6) 0.16 44 0.9991 (13) V2O5$nH2O Ads (5) 0.025 17 0.9983 (16) Cos (5) 0.025 7.8 0.9990 (18) V2O523 Cos (6) — 4.9 FeCp2 d –V2O5 24 Cos (7) 0.8 0.46 ZrO247 Cos (7) 1.42 TiO2 48 Cos (7) — SiO248 Cos (7) 17.6

ZrO2/chitosan49 Ads (6.5) 0.89

TiO2/chitosan50 Ads (7.2) 9.25

ZrO2/Nafion47 Cos (7) 48

TiO2/Nafion48 Cos (7) 105.7

SiO2/Nafion48 Cos (7) 71.7

aImm¼ immobilization, Ads ¼ adsorption, and Cos ¼ cosedimentation.bQ¼ mass ratio of G ¼ GOx and V ¼ V

2O5or K2[V6O16].cCalculated from

the slope of the linear part of the calibration curve.dFeCp2¼ ferrocene.

and TMA+(tetramethylammonium)) and divalent cations

(M2+¼ Ca2+, Mg2+and Ba2+). All these phases have been fully

characterized by combining different techniques (i.e. X-ray diffraction, TGA and chemical analysis, UV-vis spectroscopy,

51V MAS NMR spectroscopy and SEM) in order to obtain

a precise description of their structure which should be of great benefit when using them in more complex materials such as sensors. The experimental conditions (initial pH and ageing time) and chemical composition of M[V3O8] and Mx[V6O16] (x¼ 1 and 2) phases are gathered in Table 1(b). The XRD diffraction patterns of solids obtained at pHi> 2 show that they correspond to trivanadate or hexavanadate phases with various amounts of intercalated water (see Table 1(b), Fig. 2 for M+¼ K+and S1 of

ESI†). The molar ratio M/V of these compounds is confirmed by chemical analysis. The amount of water is determined by TGA analysis. The vanadium oxides obtained with Na+, Mg2+, Ca2+

and Ba2+ ions are isostructural and this framework can be

described as a hewettite type structure (Fig. 1(a)) which is made up of layers with VO5trigonal bipyramid chains and distorted VO6octahedral chains sharing corners.31Vanadium oxide phases with K+, Cs+and NH

4+ions exhibit a different vanadium-oxide framework (Fig. 1(b)) made of twisted zigzag chains composed of VO5 square pyramids sharing corners and edges and distorted VO6octahedra.32It is worth noting that the geometry of VO6and VO5 polyhedra in these structure types is close. In particular, there is only a slight difference between the angular conforma-tions of a trigonal bipyramid and a square pyramid.32bIn both structures, cations are intercalated between the layers. The hex-avanadate phase of a specific cation (Cs+, NH

4+and Ba2+) may present various amounts of intercalation water depending on initial pH conditions. At the SEM scale, most precipitates consist of rods, which are about 500 nm wide (see Fig. 3(c) and (d) and S2 of ESI†) and a few micrometres long. However, for the same cation, variation of the hydration rate of the vanadium oxide can strongly impact on the particle morphology. As an example, for (NH4)2[V6O16]$H2O, the SEM image shows butterfly like aggregates consisting of elongated platelet crystals which are hundreds mm long and 20 mm wide (Fig. 3(a) and (b)). In

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 A contrast, for (NH4)2[V6O16]$1.5H2O the sample is made of entangled fibres of 10 mm long and 200 nm wide (Fig. 3(c)). For K2[V6O16]$nH2O, two kinds of crystals are present in the precipitate (see Fig. 3(d), 6 and S3 of ESI†). Spherical aggregates of about 60 mm in diameter are observed that result from the assembly of rods 1–2 mm large together with hexagonal platelets of hundreds mm large (see Fig. 6, vide infra). These two kinds of crystals may correspond to the two crystalline structures of K2[V6O16]$nH2O (n ¼ 1.5 and 2.7) that were evidenced by powder XRD (Fig. 2). The local environments of vanadium in the trivanadate and hexavanadate phases were investigated by

51V MAS NMR spectroscopy. The set of 51V parameters

extracted from the simulations is listed in Table 3. For both types of structures (i.e. hewettite and K2[V6O16]), the spectra consist of two overlapping manifolds of spinning sidebands representing two resonances that correspond to the two non-equivalent vanadium sites of the asymmetric unit. As an example, Fig. 4(a) and (b) display experimental and simulated 51V MAS NMR

spectra of (NH4)2[V6O16]$H2O that present two resonances at 514 and 551 ppm. The assignment of signals is not straightforward since 51V NMR parameters of both vanadium

sites are similar in the hewettite type structure (M+ ¼ Na+ or M2+ ¼ Ca2+, Mg2+, and Ba2+) and the K

2[V6O16] structure type (M+ ¼ K+, NH

4+, and Cs+) (see Table 3). This may be explained by the fact that both vanadium sites exhibit a similar distorted geometry that can be described as a distorted square bipyramid. The coordination sphere of vanadium is composed of a short

V]O bond of about 1.6 A, four V–O bonds of about 1.7–1.9 and a long V–O bond (>2 A) in trans position of V]O. A limit of 2.6 A for V–O distances is generally assumed for including the corresponding oxo ligand in the coordination sphere of vana-dium.33 Depending on this latter bond length, the coordination polyhedron is either close to a square pyramid (for a V–O bond of 2.9 A) or to a distorted octahedron (for a V–O bond of 2.3 A). For the K2[V6O16] type structure (M+ ¼ K+, NH4+, and Cs+), one can observe that the Ddiso parameter is systematically higher for the signal close to 510 ppm (Table 3). As reported previously for a series of polyoxovanadates, this parameter can precisely probe the local distortion of vanadium sites. It was reported that

4

square pyramidal environment (Vsp). Consequently, the signal at 548 ppm may be attributed to the octahedral site of the structure (VO). This assignment is in agreement with that reported for K[V3O8].34a It is quite difficult to distinguish the hewettite and the K2[V6O16] type structures on the basis of51V NMR parameters, since only the values of the isotropic chemical shift (diso) are significantly different between the two type structures. Therefore, in this class of compounds, the isotropic chemical shift is certainly influenced by the vanadium oxide framework and the connectivity between vanadium polyhedra. The K2[V6O16] type structures (M+¼ K+, NH+, and Cs+) are characterised by a first signal at520 < diso<510 ppm and a second at551 < diso<536 ppm whose difference of disois close to 30–40 ppm (except for Cs2[V6O16]$0.7H2O). In contrast for the hewettite type structure (M+¼ Na+or M2+¼ Ca2+, Mg2+,

and Ba2+), both signals are systematically lower than520 ppm

(i.e. one signal at547 < diso <535 ppm and a second one at566 < diso <548 ppm) and their difference is reduced to approximately10–20 ppm. The resolution of the 51V spectra

seems to depend strongly on the crystallinity and the hydration rate of vanadium oxides. For the hydrated phases (i.e. K2[V6O16]$nH2O (n ¼ 1.5 and 2.7), Mg[V6O16]$7.1H2O, Ca [V6O16]$9.7H2O and Ba1,2[V6O16]$5.5H2O), two very broad signals are present that merge into one single broad resonance in the isotropic part of the spectrum (see Fig. 5). In contrast for Cs [V3O8] and (NH4)2[V6O16]$H2O, the isotropic part of the spec-trum is composed of two resolved sharp signals. The broadening of the lines possibly results from a distribution of VOxsites with different local environment (coordinated water molecules, hydrogen bonds,.) as previously evidenced for Cs2[V6O16]$ 0.7H2O.27

To conclude, we have shown that51V NMR parameters of the

hewettite and K2[V6O16] structure types are quite close, making difficult the identification of both structures from the NMR point of view. In particular, the similarity of quadrupolar and the |DdCSA| parameter is typically lower than 320 ppm for VO4 units in ortho-, pyro- and metavanadates while it is larger than 260 ppm for VO5units in divalent metal metavanadates.34 DdCSA parameters are particularly large for strongly distorted VO6 units in V2O5$nH2O xerogels ($500 ppm)26,35 or decavanadate poly-anions.36 Therefore, for (NH4)2[V6O16]$H2O, the signal at 514 ppm with the larger DdCSA parameter (480 ppm) may be assigned to the more distorted vanadium site i.e. the site with the

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Table 3 Isotropic chemical shift (diso) a

, quadrupole coupling constant (CQ), quadrupolar asymmetry parameter (hQ), chemical shift anisotropy

(DdCSA) a

and CSA asymmetry parameter (hCSA) a

obtained by QUASAR simulationsbof51V MAS NMR spectra

Vanadium oxide diso (ppm) CQ(kHz) hQ DdCSA (ppm) hCSA Ref.c K2[V6O16]$2.7H2O d 548 2450 0.44 405 0 tw 510 3030 0.89 459 0 Cs[V3O8] 548 3000 0.6 405 0.1 27 517 3000 0.7 460 0.2 Cs[V6O16]$0.7H2O 536 2200 0.7 400 0.2 27 520 3000 0.8 460 0.1 (NH4)2[V6O16]$nH2O n¼ 1 and 1.5e 551514 27003000 0.30.8 420480 0.10.1 tw Na[V3O8]$1.5H2O 548 3100 0.9 420 0 26 535 2400 0.8 370 0 Mg[V6O16]$7.1H2O 549 3100 0.9 420 0 tw 538 2400 0.8 420 0 Ca[V6O16]$9.7H2O 553 2800 0.7 370 0.1 tw 535 3100 0.4 400 0.1 Ba[V6O16]$5.5H2O 566 3000 0.9 420 0.1 tw 547 3000 0.9 320 0.2 a

Chemical shift tensor parameters diso,DdCSAand hCSAare defined as

diso¼ (d11+ d22+ d33)/3,DdCSA¼ (d33 diso) and hCSA¼ (d22 d11)/

(d33 diso).bThe data were obtained with the following accuracy : CQ

 0.1 MHz; hQand hCSA  0.1; diso  1 ppm; DdCSA  10 ppm;

(4, c, j) close to (0, 60, 0) values.cReferences; tw

¼ this work.dFor

this spectrum, only K2[V6O16]$2.7H2O was characterized. e

For n¼ 1 or 1.5, the spectra are almost identical.

CSA parameters between both structure types is consistent with the presence of vanadium sites of similar local environment (strongly distorted VO5geometry). Only the isotropic chemical shift parameter with notably different values for both structure types seems to be more sensitive to the vanadium oxide framework.

3.2 Kinetics of transformation of MV2O5intercalates into M[V3O8] (or M2[V6O16]) phases depending on the nature of intercalated cation M+(M2+)

In order to select the best candidate for enzyme immobilization, the different tri- and hexavanadates were compared in terms of their kinetics of formation and pH stability range. The synthesis of trivanadates and hexavanadates is based on the preparation of V2O5$nH2O gels at pHz 1.5. By adding at constant pH small amounts of cations to an acidic vanadate solution of pH 1, MxV2O5intercalates are first obtained (Table 1(a)). The cation content (x  0.3 for M+and x  0.15 for M2+) (Table 1(a))

obtained by elemental analysis was typically observed for the already prepared V2O5$1.8H2O xerogel films in which mono-valent cations were intercalated.37,38 By increasing pH above 1 with MOH or M(OH)2solutions, precipitation of M[V3O8] and Mx[V6O16] (x¼ 1 and 2) phases occurs. Depending on the nature of cation, these vanadium oxides precipitate directly from the vanadate solution or result from the progressive transformation of MI

0.3V2O5(or MII0.15V2O5) intercalates.26In the first case, the tri- and hexavanadate phases are formed in a short time (1 day) and at very acidic pH (pH 1–2). In the second case, precipitation occurs at higher pH (3–4) and after at least 4 days (Table 1).

The kinetics of formation and thermodynamic stability of the tri- and hexavanadates seem to increase with the ionic radius of interlayer cations (see Table 1 and Fig. S4 of ESI†). Indeed, with the same experimental conditions (pHi¼ 2, 40C), the vanadate phase is fully formed within 21 days with Na+while only 1 or 4

days are respectively required with K+or NH

4+(see Table 1(b)). In fact, three behaviours can be distinguished. For small cations like Mg2+, Na+, and Ca2+ (r

ion < 1 A), the formation of tri-vanadates or hexatri-vanadates takes at least 4 days at pH 3–4. For larger cations like K+and NH

4+(1 < rion< 1.5 A), M[V3O8] or Mx[V6O16] (x¼ 1 and 2) can be obtained immediately after one day in the pH range 2–4. Finally for large cations such as Cs+,

TMA+, and Ba2+(r

ion> 1.35 A), a competition is clearly observed between the formation of M[V3O8] (or Mx[V6O16]) and that of the decavanadate polyanion. Indeed, it is known that crystalli-zation of polyoxometallates usually requires large cations to occur.39,40 The kinetics of tri- and hexavanadates formation is

particularly low in the pH range 3–4 where the decavanadate is the most abundant vanadate in solution. As an example, cesium tri- or hexavanadate is either obtained after one day at pH 1 or 21 days at pH 3. Therefore, as soon as decavanadate crystalline phases are formed, their dissociation and conversion into tri- or hexavanadate phases are not kinetically or thermodynamically favoured. In the case of the largest TMA+ cation, it is worth

noting that no tri- or hexavanadate phase could be obtained in the pH range 1–4, while precipitation of 4TMA[H2V10O28]$ 4H2O occurs.39

The influence of the cation size on the formation of tri- and hexavanadate phases is possibly related to its hydration rate.

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Actually, it has been shown previously that the water content of MxV2O5 intercalates strongly depends on the size and charge of intercalated cations.41 For monovalent cations, d

001 of ca. 10.9–11.6 A typically corresponds to intercalates with one water layer while for divalent cations, the interlamellar space contains two water layers (d001ca. 13.4–14.2 A).41An empirical relation-ship was established showing that the hydration of cations (i.e. hydration number) is proportional to the ionic charge to radius ratio Z/r parameter.41Similarly, crystallization of hexavanadate

phases of Cs+and NH

4+ions is obtained respectively with no or only one water molecule per formula unit. In contrast, at least three water molecules per V6O162 unit are required in the hexavanadate structure for smaller cations like Ba2+, K+, Mg2+or

Ca2+. However the hydration rate of cation does not seem to

influence strongly the kinetics of the M[V3O8] (or M2[V6O16] and M0[V6O16]) formation. As an example, Ca[V6O16]$9.7H2O and Na[V3O8]$1.5H2O phases are both obtained after 4 days at pH 3 and 4 while the interlayer cations Ca2+and Na+have different

hydration numbers (i.e. 11.0 and 3.4 respectively).41In contrast,

the kinetics of the M[V3O8] (or M2[V6O16] and M0[V6O16]) formation seems to be related to the crystallographic structure of the tri- and hexavanadate phases. Indeed, the formation of tri- and hexavanadate phases with the hewettite structural type (Li+, Na+, Ca2+, and Mg2+) is systematically delayed compared to

those with the K2[V6O16] type structure (K+, Cs+, and NH4+). Since neither the MxV2O5 nor the decavanadate polyanion presents a structural relationship with the M[V3O8] (or M2[V6O16]) structures, the formation of these vanadium oxides presumably implies a redissolution–precipitation process whose kinetics are dependent on the size of the cation. It has been previously reported that V2O5 gels result from the poly-condensation of a neutral precursor [VO(OH)3(OH2)2]0.22aSince the composition of the V(V) solution changes with increasing pH, a vanadate solution of pH > 1 presumably contains a mixture of a neutral species [VO(OH)3(OH2)2]0 and a negatively charged precursor [VO(OH)4(OH2)] as previously reported for the formation of Na[V3O8]$1.5H2O phases.26 Both vanadate precursors may be involved in the formation of trivanadate and hexavanadate phases.

3.3 K2[V6O16]$nH2O–GOx biosensors: design, stability and performances

Among the different trivanadates and hexavanadates reported in this work, K2[V6O16]$nH2O was selected as an immobilization matrix for enzymes since this phase is obtained in a short time (1 day), with a good yield (see Fig. S4†) and in conditions of moderate acidity (pH > 2). Moreover, since this phase can be obtained at pH close to 4, it may reasonably be expected that it is still stable at 5 < pH < 6, being therefore compatible with a number of enzymes whose activity is optimum, or at least well-preserved, in slightly acidic conditions.

In a first step, we have evaluated the stability of K2[V6O16]$ nH2O (n ¼ 1.5 and 2.7) precipitates in aqueous solutions of moderate acidity (typically between pH 3 and 6) in comparison to V2O5$nH2O gels. Preformed V2O5films on a glass support (see ESI† for experimental details) and K2[V6O16] precipitates were immersed into acetate buffers of pH 3.5, 4 and 5 and phosphate buffer of pH 6 for about 48 hours. For pH between 3.5 and 5, the

colour of V2O5 films did not change and these films were not dissociated in solution. Their XRD diagrams (see Fig. S5†) are almost identical to that of a V2O5 film at pH 1–2, showing a preservation of the bilayer structure of V2O5$nH2O gels. For pH > 5, the V2O5 films evolved rapidly to green amorphous compounds that are dissociated in solution upon ageing. In contrast, the K2[V6O16] phase did not evolve in terms of colour and quantity upon ageing in acetate or phosphate buffers at 3.5 < pH < 6 for 48 h. The stability of K2[V6O16] at pH 5 and 6 was confirmed by recording the XRD pattern (see Fig. 2) and 51V

MAS NMR spectra (see Fig. 5), both being identical to those of the K2[V6O16]$nH2O reference phase at pH 3.

In the second step, we have selected glucose oxidase as a model enzyme for immobilization studies, based on the fact that its optimal activity is close to pH 5.5.5The immobilization of GOx

has been performed through a two step adsorption and co-sedimentation methods (see Table 2 and Experimental section) both in K2[V6O16]- and V2O5$nH2O gels for comparison purposes. In both cases, homogeneous thin films of bionano-composites were formed either on glass or Pt electrodes, as shown in Fig. 6(A) for K2[V6O16]–GOx. XRD and SEM images of GOx-V2O5films are consistent with a disorganized assembly of V2O5ribbons and GOx, as a result of the enzyme addition to the colloidal solutions of V2O5 (see Fig. S5 of ESI†) and in agreement with a previous study.23 The XRD pattern of

K2[V6O16] based biomembranes (Fig. 2(b) and (c)) shows that the structure of K2[V6O16] is preserved after GOx immobilization by adsorption and co-sedimentation methods. A slight shift in the 00l series of reflections is consistent with a slight modification of the interlamellar space and indicates that the enzyme is not intercalated between the vanadium oxide layers. The SEM images of K2[V6O16]–GOx biomembrane (Fig. 6B(a)–(c)) prepared by adsorption show the coexistence of large platelets a few tens mm large and spherical assemblies of rods about 1–2mm wide. The SEM image of K2[V6O16]–GOx biomembranes (Fig. 6B(d)) prepared by co-sedimentation shows a disorganized assembly of both kinds of crystallites.

These K2[V6O16]–GOxcos were studied by IR spectroscopy, a powerful technique for the determination of the secondary structure of proteins. Indeed, the preservation of the secondary structure of GOx after immobilization in K2[V6O16] is crucial in biosensor design since conformational changes can strongly affect the enzyme catalytic activity.42The position and relative intensity of main IR absorption peaks for K2[V6O16]–GOx are listed in Table 4 and compared to those of K2[V6O16] and GOx native phases according to the representative FT-IR spectra of the samples (Fig. 7 and S6†).26,42 The spectrum of

K2[V6O16]–GOxcos presents characteristic vibration modes of GOx and K2[V6O16]. It has been previously reported that information on the secondary structure of GOx (i.e. helices, sheets, coils and turn motifs) can be obtained by analysing the vibration modes of the amide group of the peptide bond (amide I and II bands).42 For K

2[V6O16]–GOxcos, the main peptide absorption bands at 1648 cm1(n C]O amide I) and 1547 cm1 (d N–H amide II) are quite close to those of the native GOx (i.e. 1652 cm1 and 1540 cm1 respectively). The position and the

relative intensity of these vibration bands in the FT-IR spectrum of K2[V6O16]–GOxcosare consistent with the assignment of both vibration modes, suggesting that the secondary structure and

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consequently the bioactivity of GOx after immobilization in the vanadium oxide are preserved. Moreover, the FT-IR spectrum of K2[V6O16]–GOxcosshows other characteristic vibrational modes of GOx (i.e. amide III and carbon skeleton vibrations in the 1000–1500 cm1 range) (see Table 4). Finally, the FT-IR spectra of K2[V6O16]–GOxcos and K2[V6O16] exhibit typical absorption peaks of V–O stretching modes at close wavenumber positions, suggesting that the vanadium oxide skeleton is preserved after association with GOx.

After the immobilization of GOx in both K2[V6O16]$nH2O (n ¼ 1.5 and 2.7) and V2O5$nH2O, the colour of the bio-hybrid membranes turns brown, revealing a slight reduction of V5+ to V4 + for V

2O5 and K2[V6O16]$nH2O. The surface [V5+]/[V4+] composition has been determined by X-ray photoelectron spec-troscopy. Fig. S7 and S8† display the V 2p, K2p and C1s XPS spectra of the K2[V6O16] reference phase at pH 3 (i.e. K2[V6O16] – pH 3), the K2[V6O16] phase obtained after immer-sion in a phosphate buffer solution of pH 6 for 24 h (i.e. K2[V6O16] – pH 6) and K2[V6O16]–GOxcos. The K2p XPS spectra of the three samples can be fully superimposed revealing a similar K+ environment in the three compounds. The V2p3/2 spectra of the three compounds are quite similar and display a broad peak that can be fitted with two components close to 515.8 and 517.0 eV that can be assigned respectively to V4+ and V5+ species.43 The amount of V4+ and V5+ surface species could be extracted from the peak area ratios (see Table S1†) and a trend of the [V5+]/[V4+]

ratio evolution can be given. According to these

Table 4 Main peaks in the FT-IR spectra of K2[V6O16]–GOxcos, K2[V6O16] and GOx samples and corresponding assignments a

GOx K2[V6O16]$nH2O K2[V6O16]–GOxcos

Assignments

Wavenumber cm1(intensity) Wavenumber cm1(intensity) Wavenumber cm1(intensity)

3539 (sh) n O–H$(H2O) 3406 (br) 3402 (br) n O–H$(H2O) 3285 (br) n N–H (amide) 3065 (vw) 2961 (w) 2957 (w) n C–H 2929 (w) 2929 (w) n C–H 2872 (w) 2878 (w) n C–H 1645 (s) d H–O–H$(H2O) 1652 (s) 1648 (s) n C]O (amide I) 1540 (m) 1547 (m) d N–H (amide II) 1452 (w) 1458 (w) 1391 (w) 1397 (w) 1344 (w) 1347 (w) 1302 (w) 1302 (w) 1245 (w) 1251 (w) (Amide III) 1086 (br) 1096 (br) 953 (vw) 1004 (w) 1000 (m) n V]O 969 (w) 965 (m) n V]O 950 (w) 953 (m) n V]O 826 (vw) 729 (w) 737 (m) n O–V–O 657 (w) 660 (m) n O–V–O 520 (w) 523 (m) n O–V–O

abr¼ broad, m ¼ medium, sh ¼ shoulder, s ¼ strong, w ¼ weak, and vw ¼ very weak.

results, the reduction of V5+ by mixing K

2[V6O16] in a phosphate buffer at pH 6 is not significant. However, the encapsulation of GOx in K2[V6O16] leads presumably to a more significant reduction of V5+ since the [V5+]/[V4+] composition evolves roughly

from 19 to 12. The reduction of vanadium(V) to vanadium(IV) can enhance the electronic conductivity of vanadium oxides due to the electron hopping between V5+ and V4+. Therefore, the

biosensor response may take advantage of the electronic proper-ties of vanadium oxides that may enhance the rate of electron transfer, thereby increasing the intensity of the electrical signal and decreasing the detection limit of the analytes.

Finally, K2[V6O16]–GOx films were evaluated as biosensors for glucose detection without mediator using amperometric titration. Glucose oxidase (GOx) catalyzes the aerobic oxidation of glucose with concomitant production of H2O2:

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Glucose + O2¼ gluconolactone + H2O2

A significant activity of GOx (by measuring the consumption of O2) was detected in those biomembranes soaked in 50 mmol L1 glucose solutions at 3.5 < pH < 5 for V2O5and 3.5 < pH < 6 for K2[V6O16], demonstrating the possibility of sensing analytes in a large pH range. After 24 h of storage at 4C, the bioelectrodes still present a GOx activity. These preliminary experiments show that vanadium oxides can serve as a host matrix for GOx encap-sulation and preserve the biocatalytic activity at pH between 3.5 and 6. The amperometric titration of glucose was therefore per-formed by using V2O5$nH2O gels and K2[V6O16] matrices in acetate (pH 5) or phosphate buffer (pH 6) solutions. These pH values were chosen since they are close to the optimum pH of native GOx, namely pH 6.0 and in the pH stability range of the studied vanadium oxides. In order to determine the best config-uration for the glucose determination, two different amounts (50 and 100mg) of GOx were immobilized by the adsorption method. Since the biosensor response was not improved using the higher amount, the quantity of GOx was fixed at 50 mg in all bio-membranes. The electrochemical responses to successive addi-tions of glucose were recorded with rotating disk Pt electrodes at 0.7 V/Ag/AgCl which is in the potential range of hydrogen peroxide oxidation. Typical calibration curves of V2O5 and K2[V6O16] based on steady state measurement are depicted in Fig. 8. For both V2O5$nH2O gels and K2[V6O16] matrices, the bioelectrodes present rapid (t ¼ 15 s), stable and sensitive responses to glucose additions whatever the preparation method (adsorption and co-sedimentation). Both types of bioelectrodes provide an excellent linear relationship to glucose concentration

with a dynamic concentration range between 2.5 105M and 1.5 103M or 3.0 103M for K

2[V6O16] and V2O5$nH2O gels, respectively. The sensitivities are calculated from the slope of the linear part of these calibration curves (Table 2). The comparison of the sensitivities of both matrices at pH 5 shows that the performance of K2[V6O16] is superior to V2O5$nH2O. Moreover, immobilisation of GOx by co-sedimentation with V2O5caused a strong decrease of the sensitivity value presumably due to a partial denaturation of enzymes when mixed with this matrix. K2[V6O16] appears to be more biocompatible. The same high value of sensitivity (44–48 mM M1 cm2) is obtained with K2[V6O16] at pH 6 whatever the immobilization method, in agreement with a better preservation of GOx bioactivity in phosphate buffer at pH 6 compared to that in acetate buffer at pH 5 for which the sensitivity is twice as low. The same tendency is observed with the maximum current densities at saturating glucose conditions (Jmax) which reflects the amount of immobi-lized proteins exhibiting an electro-enzymatic activity. Indeed the value of Jmaxis two times lower at pH 5 (42mA cm2) than at pH 6 (88–78mA cm2).

In order to estimate the repeatability, the reproducibility and the storage stability of our glucose biosensor, we have selected the K2[V6O16]–GOxcosbiosensor at pH 6. The repeatability of response current was investigated for ten successive additions of 10 mM glucose (Fig. 8(C)). The relative standard deviation coefficient (RSD) was 6%. The detection limit determined for a signal to noise ratio of 3 was 5 mM. Four electrodes, made independently, showed a reproducibility of 6.5%. Two K2[V6O16]–GOxcos electrodes were stored in phosphate buffer solution (pH 6) at 4C to study their storage stability for four weeks. The biosensor responses as a function of storage time correspond to an average value of 5 measurements (10 mM glucose). The biosensor responses were stable for one week, afterwards a slow decrease of the signal was observed. After four weeks, the biosensor responses were around 40% of their initial value (Fig. S9†).

To the best of our knowledge, there are only two other reports dealing with V2O5–GOx based biosensors in the literature, involving either thin films of reduced V2O523or nanocomposites based on V2O5$nH2O gels intercalated by ferrocene (FeCp2) and mixed with a polymer matrix (PVA).24 Ferrocene serves as

a reducing agent of V(V) as well as a redox mediator for the enzyme regeneration. Compared to the pure mediator system (FeCp2/PVA),24 the biosensor displays 20% increase of sensi-tivity. In both previous studies, the biosensor performance is promising and is certainly due to the electrical conductivity of V2O5. In particular, the vanadium oxide framework can establish the electron communication between the electrode surface and FeCp2 or GOx, thereby enhancing the electrical signal of the transducer and leading to an improved sensitivity and a lower detection limit of analytes. In this context, the performance of the here-described K2[V6O16]–GOx membranes was compared to those calculated for other biosensors using V2O523,24(Table 2). The sensitivities obtained in the present work are significantly higher than those reported in these two systems, showing that K2[V6O16]$nH2O (n¼ 1.5 and 2.7) is a more suitable matrix for enzyme encapsulation than V2O5. Similarly, these biosensor performances can also be compared to those reported for other glucose biosensors based on metal oxide/Pt electrodes (Table 2).

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The sensitivities of the present biosensors are higher than other GOx biosensors based on sol–gel metal oxides (ZrO2 and SiO2) (see Table 2). Since cracks and fractures are often observed in dry monolithic sol–gel metal oxide films, composite electrodes can be alternatively prepared by adding organic polymers (chitosan or Nafion) into sol–gel solutions. The use of Nafion appears to be particularly efficient, with the best sensitivities for TiO2and SiO2. In the present work, the vanadium oxide films are quite homo-geneous and stabilization by adding another organic component is not necessary, giving rise to quite good sensitivity, detection limit and storage stability.

4.

Conclusion

There is currently a strong demand for functional materials that can be formed and/or can operate under conditions compatible with the preservation of biological activities. In terms of chem-istry, these conditions, i.e. moderate temperature, near neutral pH, no organic solvents, and toxicity, are binding enough to disqualify a number of well-known materials. Therefore, there is a need to explore more widely the diversity of materials composition and structures to identify suitable candidates for bionanocomposite design.

Looking for potential electroactive hosts for the building-up of enzymatic biosensors, we have shown that the room temperature process leading to the synthesis of Na[V3O8]$1.5H2O can be extended to a large range of cations including divalent ones. This chemical route leads to nanostructured tri- and hexavanadate phases with either the hewettite- or K2[V6O16] type crystalline structures. The size and morphology of crystallites depend both on the nature of cation and hydration rate of the oxides, showing the possibility of finely tuning the texture of the material by this process. We have observed that the kinetics of the formation of vanadium oxides with the K2[V6O16] type structure (i.e. K+, Cs+, and NH4+) is significantly enhanced compared to those with the hewettite-type structure (i.e. Li+, Na+, Ca2+, and Mg2+). Finally,

the nanostructured K2[V6O16]$nH2O material has been used for the first time to build up enzymatic based biosensors. The K2[V6O16] based amperometric biosensor presents superior properties to V2O5$nH2O gels in terms of sensitivity and repro-ducibility, showing that these phases may presumably compete with other inorganic host matrices through the optimization of the biosensor preparation. The good stability of K2[V6O16]$ nH2O in a large pH range and different buffers shows that this matrix is apparently tunable to a wide range of specific condi-tions (pH, buffer,.), allowing the extension of this approach to a wide range of enzymes and sensing analytes even at low pH values. Further developments are now necessary to fully take advantage of the electrocatalytic activity of K2[V6O16]$nH2O in sensing devices, including composite approaches involving biopolymers that should increase the bio-compatibility of the host material as well as widen the pH range of stability.44

Moreover, an increase of the V4+content may enhance the rate of

the electron transfer thereby increasing the sensitivity of the bioelectrodes.

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23 V. Glezer and O. Lev, J. Am. Chem. Soc., 1993, 115, 2533. 24 C. G. Tsiafoulis, A. B. Florou, P. N. Trikalitis, T. Bakas and

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25 (a) K. West, B. Zachau-Christiansen, S. Skaarup, Y. Saidi, J. Barker, I. I. Olsen, R. Pynenburg and R. Koksbang, J. Electrochem. Soc., 1996, 143, 820; (b) M. Dubarry, J. Gaubicher, D. Guyomard, N. Steunou and J. Livage, Chem. Mater., 2004, 16, 4867; (c)

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26 O. Durupthy, N. Steunou, T. Coradin, J. Maquet, C. Bonhomme and J. Livage, J. Mater. Chem., 2005, 15, 1090.

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Fig. 1 (a) Projection of the structure of Na[V3O8]$1.5H2O along [010]

and [100]. (b) Projection of the structure of Cs[V3O8] along [100] and

[001]. Interlayer cations are represented by white (Na+) and grey (Cs+)

circles.

Fig. 2 Powder XRD patterns of (a) K2[V6O16]$nH2O at pH 3,

(b) K2[V6O16]–GOx biomembrane prepared by adsorption at pH 6 and

(c) K2[V6O16]–GOx biomembrane prepared by co-sedimentation

(K2[V6O16]–GOxcos) at pH 6. (1) and (2) are respectively XRD patterns of

K2[V6O16]$2.7H2O and K2[V6O16]$1.5H2O provided by the JCPDS card

no. 51-379 and 54-0602. * corresponds to an impurity.

 A

Fig. 3 SEM images of (a) and (b) (NH4)2[V6O16]$H2O (pHi ¼ 2; ageing

time ¼ 4 days), (c) (NH4)2[V6O16]$1.5H2O (pHi¼ 3; ageing time ¼ 14

(14)

Fig. 4 (a) 51V MAS NMR spectrum of (NH

4)2[V6O16]$H2O spun at

14 kHz and recorded at 79.0 MHz with 14 000 transients and (b) simulation obtained with QUASAR.

Fig. 5 51V MAS NMR spectrum of K

2[V6O16]$nH2O spun at 14 kHz

and recorded at 79.0 MHz with 14 000 transients. The spectra were recorded after immersing K2[V6O16]$nH2O precipitates in a solution of

(a) phosphate buffer of pH 6 or (b) acetate buffer of pH 5 (* corresponds to an impurity).

Fig. 6 (A) Photographs of (a) the suspension of K2[V6O16] deposited on

a Pt electrode and (b) the dried K2[V6O16]–GOx coating prepared by

adsorption on the Pt electrode. (B) SEM images of (a), (b) and (c) K2[V6O16]–GOx biomembrane prepared by adsorption, and (d)

K2[V6O16]–GOx biomembrane prepared by co-sedimentation.

Fig. 7 Representative FT-IR spectrum of the K2[V6O16]–GOxcos

(15)

Fig. 8 Glucose calibration curves of (A) K2[V6O16]–GOx (Ads, pH 6,

curve a) and V2O5–GOx (Ads, pH 5, curve b); (B) K2[V6O16]–GOx

(Cos, pH 6, curve a) and V2O5–GOx (Cos, pH 5, curve b) (50 mg GOx,

Eapp ¼ 0.7 V/Ag/AgCl). (C) Dynamic response after ten subsequent

additions of 10 mM of glucose in phosphate buffer, pH 6 for the K2[V6O16]–GOxcosbiosensor (50 mg GOx, Eapp¼ 0.7 V/Ag/AgCl,

(16)

10 20 30 40 50

VNH

4

-2

VNH4-2-1 VNH4-2-4 VNH4-2-7 NH4-2-14 VNH4-2-21 In te n si té r el at iv e 2 theta 10 20 30 40 50

VNH4-3

VNH4-3-1 VNH4-3-4 VNH4-3-7 VNH4-3-14 VNH4-3-21 2 theta

Electronic Supplementary Information (ESI) for Journal of Materials Chemistry

A general route to nanostructured M[V

3

O

8

] and M

x

[V

6

O

16

] (x = 1 and 2)

and their first evaluation for building enzymatic third generation

biosensors.†

Nathalie Steunou,

* a,b

Christine Mousty,

c

Olivier Durupthy,

a

Cécile Roux,

a

Guillaume

Laurent,

a

Corine Simonnet-Jégat,

b

Jacky Vigneron,

b

Arnaud Etcheberry,

b

Christian

Bonhomme,

a

Jacques Livage,

a

and Thibaud Coradin.

a

Fig. S1: X-ray diffraction patterns of M[V

3

O

8

] and M[V

6

O

16

] phases. In the title of

figures VM-X-Y, M corresponds to the cation, X to the pH value and Y to the ageing

time.

(17)

10 20 30 40 50

VMg-2

VMg-2-1 VMg-2-4 VMg-2-7 VMg-2-14 VMg-2-21 2 theta 10 20 30 40 50

VCa-2

VCa-2-1 VCa-2-4 VCa-2-7 VCa-2-14 VCa-2-21 2 theta 10 20 30 40 50

VBa-2

VBa-2-1 VBa-2-4 VBa-2-7 VBa-2-14 VBa-2-21 2 theta (°)

Fig. S1: X-ray diffraction patterns of M[V

3

O

8

] and M[V

6

O

16

] phases. In the title of

figures VM-X-Y, M corresponds to the cation, X to the pH value and Y to the ageing

time.

(18)

(a)

A1

A2

(b)

B1

B2

(c)

C1

C2

Fig. S2: SEM images for (a) Mg[V

6

O

16

]7.1H

2

O (images A1 and A2, pH

i

= 2, 4 days); (b)

Ca[V

6

O

16

] 9.7H

2

O (image B1 pH

i

=2, 7 days) (image B2 pH

i

= 3, 14 days) ; (c)

(19)

0

20

40

60

80

100

0

5

10

15

20

Li+ Na+ K+ NH4+ Cs+ Mg2+ Ca2+

%

tr

i-he

xa

-v

an

a

d

at

e

s

ageing (days)

Fig S3. SEM images of K

2

[V

6

O

16

]

-GOx biomembrane prepared by adsorption.

Fig S4: Relative amount of tri- and hexavanadates (i. e. m (MV

3

O

8

/(m(MV

3

O

8

)+ m(V

2

O

5

))

versus ageing at pH 2 for different monovalent and bivalent cations. The relative amount of

Ba

1.2

[V

6

O

16

] is not reported because of the concomitant presence of 3Ba[V

10

O

28

]21H

2

O in a

(20)

Fig. S5. XRD pattern of (a) V

2

O

5

.nH

2

O gel at pH 1, (b) V

2

O

5

.nH

2

O gel at pH 4 and 5.

For these experiments, V

2

O

5

.nH

2

O gels are deposited on glass substrates and the resulting

films are immersed in acetate buffers of pH between 3 and 5. Due to the preferential

orientation of V

2

O

5

ribbon particles on flat surfaces, the XRD pattern of V

2

O

5

.nH

2

O gels

at pH between 1 and 5 display only the series of 00ℓ reflections. The XRD diagrams of

V

2

O

5

.nH

2

O gels at 3<pH<5 indicate that the structure of the gels is preserved until pH 5

despite of a little difference in the d

001

value indicating a slight modification of the

interlamellar space and the presence of an impurity of small amount at pH 5 (*).

The XRD pattern of V

2

O

5

sol at pH 5 and V

2

O

5

.nH

2

O-GOx biomembranes do not display

XRD reflections. Actually, by redispersion of the gel in aqueous solution, the resulting V

2

O

5

sol is then deposited on the glass substrate and dried. The stacking of V

2

O

5

particles is not

anymore present in the dried V

2

O

5

film and the particles are completely disorganized. Since

V

2

O

5

-GOx biomembrane results from the adsorption of enzyme onto these V

2

O

5

films, no

XRD reflections are present in the pattern of the V

2

O

5

.nH

2

O-GOx biomembranes.

a

b

5 10 15 20 25 30 35 40 45 50 2-théta (°) pH 4 pH 5 001 003 004 005 5 10 15 20 25 30 35 40 45 50 2-theta (°)

(001)

(003)

(004) (005)

b

*

*

(21)

**K2V6O16 ATR 520 657 729 950 969 1004 1645 3406 0 10 20 30 40 50 60 70 80 90 100 1000 2000 3000 Wavenumber (cm-1) % T GOX 826 953 1086 1245 1302 1344 1391 1452 1540 1652 2872 2929 2961 3065 3285 40 50 60 70 80 90 100 110 1000 2000 3000 Wavenumber (cm-1) % T

Fig. S6. Representative FT-IR spectra of pure a) K

2

[V

6

O

16

].nH

2

O and b) GOx samples.

(a)

(a)

(22)

Fig. S7. V2p3/2 XPS spectra of a) K

2

[V

6

O

16

]- pH 3, b) K

2

[V

6

O

16

]- pH 6) c) K

2

[V

6

O

16

]-GOx

cos

(a)

(b)

(23)

Fig. S8. Comparison of a) the V2p , b) K2p and C1s XPS spectra for K

2

[V

6

O

16

]-pH 3,

(24)

Table S1. V2p 3/2 peak positions and atomic ratio for K

2

[V

6

O

16

]-pH 3, K

2

[V

6

O

16

]-pH 6) and

K

2

[V

6

O

16

]-GOx

cos

sample

Positions

(eV)

Assignment

FWHM

(eV)

Area (P)

CPS.eV

% [V

5+

]/[V

4+

]

K

2

[V

6

O

16

]-pH 3

515.79

516.93

V2p Scan A (V

4+

)

V2p (V

5+

)

1.21

1.21

2028.43

39416.73

0.27

5.24

19.4

K

2

[V

6

O

16

]-pH 6

515.89

517.07

V2p Scan A (V

4+

)

V2p3 (V

5+

)

1.28

1.28

767.73

8769.83

0.13

2.17

16.7

K

2

[V

6

O

16

]-GOx

cos

515.77

516.96

V2p 3A (V

4+

)

V2p3 (V

5+

)

1.27

1.27

653.64

8519.45

0.11

1.38

12.5

Time (days)

Fig. S9. Storage stability of the K

2

[V

6

O

16

]-GOx

cos

biosensor (pH 6.0)

0

20

40

60

80

100

120

0

1

0

2

0

30

R

e

spo

nse

 (%)

Figure

Table 1 Experimental conditions (pH i and ageing time), chemical composition of (a) M x V 2 O 5 $ nH 2 O and (b) M[V 3 O 8 ], M x [V 6 O 16 ] (x ¼ 1 and 2) phases
Table 2 Amperometric responses to glucose of different metal oxide based biomembranes
Table 3 Isotropic chemical shift (d iso ) a , quadrupole coupling constant (C Q ), quadrupolar asymmetry parameter ( h Q ), chemical shift anisotropy ( Dd CSA ) a and CSA asymmetry parameter (h CSA ) a obtained by QUASAR simulations b of 51 V MAS NMR spect
Table 4 Main peaks in the FT-IR spectra of K 2 [V 6 O 16 ]–GOx cos , K 2 [V 6 O 16 ] and GOx samples and corresponding assignments a
+7

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